A long-standing hypothesis is that changes in regulation play an important role in adaptive evolution, notably in primates. Consistent with this theory, the past decade of research has yielded an increasing number of cases where regulatory changes contribute to species-specific adaptations and to reproductive isolation. Nonetheless, to date, there are still only a handful of examples of regulatory adaptations in primates and even fewer cases where the underlying regulatory mechanisms have been resolved. In this application, we propose to continue our work towards understanding the evolutionary processes that shape gene regulation in primates, and in particular, to study the mechanisms of regulatory change in humans and close evolutionary relatives. By using RNA sequencing instead of traditional microarrays, we propose to study and compare gene expression phenotypes in multiple tissues and across species, at unprecedented resolution, as well as to characterize exon usage and alternative splicing patterns. Moreover, by using a combination of genomic approaches that will allow us to characterize histone modification marks and methylation profiles at genome-wide scale, we propose to move beyond simple characterization of gene expression levels to the study the underlying regulatory mechanisms such as chromatin state and epigenetic markers. Specifically, we propose to: (i) Use RNA sequencing to identify genes whose transcript regulation likely evolves under natural selection, (ii) compare promoter methylation states across tissues and species in order to increase our understanding of the mechanisms of regulatory change, and (iii) compare profiles of two histone modifications across tissues and species in order to increase our understanding of the mechanisms of regulatory change. At the conclusion of this work, we will have high-resolution gene and exon expression data, methylation state, and histone modification profiles from a set of five tissues from multiple human, chimpanzee, and rhesus macaque individuals. These data will allow us to explore conserved inter-tissue regulatory differences in the three species, as well as identify genes and pathways whose regulation evolved under natural selection in primates, including human-specific regulatory adaptations. In addition, our data will allow us to determine the mechanisms that explain, at least in part, regulatory differences between the species.